Liquid chromatography high‐resolution mass spectrometry for fatty acid profiling

Quantification of fatty acids has been crucial to elucidate lipid biosynthesis pathways in plants. To date, fatty acid identification and quantification has relied mainly on gas chromatography (GC) coupled to flame ionization detection (FID) or mass spectrometry (MS), which requires the derivatization of samples and the use of chemical standards for annotation. Here we present an alternative method based on a simple procedure for the hydrolysis of lipids, so that fatty acids can be quantified by liquid chromatography mass spectrometry (LC‐MS) analysis. Proper peak annotation of the fatty acids in the LC‐MS‐based methods has been achieved by LC‐MS measurements of authentic standard compounds and elemental formula annotation supported by ¹³C isotope‐labeled Arabidopsis. As a proof of concept, we have compared the analysis by LC‐MS and GC‐FID of two previously characterized Arabidopsis thaliana knock‐out mutants for FAD6 and FAD7 desaturase genes. These results are discussed in light of lipidomic profiles obtained from the same samples. In addition, we performed untargeted LC‐MS analysis to determine the fatty acid content of two diatom species. Our results indicate that both LC‐MS and GC‐FID analyses are comparable, but that because of higher sensitivity and selectivity the LC‐MS‐based method allows for a broader coverage and determination of novel fatty acids.     

Heat shock‐induced metabolic conversion of membrane lipids,fatty acids and volatile organic compounds of Pyropia haitanensis under different heat shock time

Lipid metabolites play an important role in understanding the stress physiology of Pyropia haitanensis, and can be used to facilitate development of stress‐resistant Pyropia cultivars. Therefore, in this study ultra performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC‐Q‐TOF‐MS) and gas chromatography–mass spectrometry (GC–MS) based metabolomics approaches were developed to screen the responses of lipid metabolites such as phospholipids, glycolipids, fatty acids and volatile organic compounds (VOCs) to different heat shock times. A total of 26 potential lipid biomarkers including Lyso‐monogalactosyldiacylglycerol (Lyso‐MGDG), Lyso‐digalactosyldiacylglycerol (Lyso‐DGDG), sulfoquinovosylmonoacylglycerols (SQMG), sulfoquinovosyldiacylglycerol (SQDG), diacylglyceryltrimethylhomoserine (DGTS), triacylglycerol (TAG), Lyso‐phosphatidicacid (Lyso‐PA), Lyso‐phosphatidylcholine (Lyso‐PC), Lyso‐phosphatidylethanolamine (Lyso‐PE), Lyso‐phosphatidylglycerol (Lyso‐PG), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylinositol phosphate (PIP) were identified, most of which responded to high temperature by reducing or increasing levels after stimulation for 1 h or 6 h. After times longer than 6 h, the levels of most lipids gradually recovered to the control group levels. Moreover, the balance of lipids and fatty acids transformation was disrupted. Overall, 11 total fatty acids (TFAs), 13 free fatty acids (FFAs) and 29 VOCs were identified during 0–72 h of high temperature stress. The FFAs, especially polyunsaturated C 20 fatty acids and VOCs, showed opposing change trends, indicating the transformation between C 20 fatty acids and VOCs. Overall, this study provides important insights into the metabolic variations of P. haitanensis under different heat shock time and the relationship between the conversion of lipids, fatty acids, and VOCs. The information provided herein will facilitate efficient development and improvement of Pyropia quality by producing cultivars resistant to high temperature.     

High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids.

A sensitive negative chemical ionization (NCI) gas chromatography-mass spectrometry (GC-MS) method for the detection of pentafluorobenzyl (PFB) esters of deuterated fatty acids is described. Deuterated linoleic [18:2n-6 2H4-9,10,12,13] and linolenic [18:3n-3 2H5-17,17,18,18,18] acids were converted to chain-elongated and desaturated products during incubations with homogenates prepared from rat liver. The extracted fatty acids were derivatized with pentafluorobenzyl bromide and analyzed in the negative ion mode by GC-MS. The detection limit of the PFB esters in NCI using selected ion monitoring was below 10 femtograms. In general, detection of the PFB derivatives using the negative ion mode was more than three orders of magnitude more sensitive than using a positive chemical ionization (PCI) method with methyl ester derivatives. The PFB esters of the 2H4-18:2n-6 metabolites eluted with their unlabeled analogues, whereas the PFB esters of the 2H5-18:3n-3 metabolites were resolved from the unlabeled compounds on polar capillary FFAP columns. Isotope ratios of the 2H4-18:2n-6 metabolites were used to quantify the deuterated compounds from standard dilution curves generated from the ion abundances of the unlabeled fatty acids. The 2H5-18:3n-3 metabolites were quantified similarly using 18:3n-3. This method is feasible for the study of the in vivo metabolism of deuterated essential fatty acids in whole animals.     

Revealing the polar lipidome,pigment profiles,and antioxidant activity of the giant unicellular green alga,Acetabularia acetabulum

Marine algae are one of the most important sources of high-value compounds such as polar lipids, omega-3 fatty acids, photosynthetic pigments, or secondary metabolites with interesting features for different niche markets. Acetabularia acetabulum is a macroscopic green single-celled alga, with a single nucleus hosted in the rhizoid. This alga is one of the most studied dasycladalean species and represents an important model system in cell biology studies. However, its lipidome and pigment profile have been overlooked. Total lipid extracts were analyzed using hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS), tandem mass spectrometry (MS/MS), and high-performance liquid chromatography (HPLC). The antioxidant capacity of lipid extracts was tested using DPPH and ABTS assays. Lipidomics identified 16 polar lipid classes, corresponding to glycolipids, betaine lipids, phospholipids, and sphingolipids, with a total of 191 lipid species, some of them recognized by their bioactivities. The most abundant polar lipids were glycolipids. Lipid classes less studied in algae were identified, such as diacylglyceryl-carboxyhydroxymethylcholine (DGCC) or hexosylceramide (HexCer). The pigment profile of A. acetabulum comprised carotenoids (17.19%), namely cis-neoxanthin, violaxanthin, lutein and β,β-carotene, and chlorophylls a and b (82.81%). A. acetabulum lipid extracts showed high antioxidant activity promoting a 50% inhibition (IC₅₀) with concentrations of 57.91 ± 1.20 μg · mL⁻¹ (438.18 ± 8.95 μmol Trolox · g⁻¹ lipid) in DPPH and 20.55 ± 0.60 μg · mL⁻¹ in ABTS assays (918.56 ± 27.55 μmol Trolox · g⁻¹ lipid). This study demonstrates the potential of A. acetabulum as a source of natural bioactive molecules and antioxidant compounds.     

Unequivocal Enantiomeric Identification and Analysis of 10 Chiral Pesticides in Fruit and Vegetables by QuEChERS Method Combined With Liquid Chromatography‐Quadruple/Linear Ion Trap Mass Spectrometry Determination

In this research, 10 chiral pesticides in fruits and vegetables were simultaneously determined using chiral liquid chromatography triple quadrupole‐linear ion trap hybrid mass spectrometry (LC‐QqLIT). The QuEChERS method was applied for sample preparation, and an enhanced product ion (EPI) scan was used to acquire tandem mass spectrometry (MS/MS) spectra for the library search. Parameters including limit of detection (LOD), limit of quantification (LOQ), linearity, relative standard deviation (RSD), and matrix effects were evaluated in five representative matrices (strawberry, leek, cowpea, tomato, and eggplant). Good linearity with coefficient of determination (r²) ≥0.997 was obtained for all 20 enantiomers in these five matrices over the range from 1.0 to 250 µg L^‐1. All the recoveries at 5 and 50 µg kg^‐1 (n = 5) ranged between 70% and 120% with RSD below 20%, indicating satisfactory precision. The LOQ for the enantiomers ranged between 0.05 and 1 µg kg^‐1. Based on the proposed method, 135 commonly consumed fruits and vegetables taken from markets in Guizhou province, China, were analyzed. Enantioselective degradation for the selected chiral pesticides was observed in most of the positive samples. Chirality 27:958–964, 2015. © 2015 Wiley Periodicals, Inc.     

广叶绣球菌不同生长阶段栽培基质中代谢物差异分析

【背景】栽培基质的利用是广叶绣球菌(Sparassis latifolia)栽培中重要的生理过程，但栽培过程中基质代谢物的变化尚不清楚。【目的】通过不同生长阶段栽培基质中差异代谢物分析挖掘关键代谢物，为广叶绣球菌基质利用机理研究提供理论参考。【方法】利用UHPLC-MS/MS技术分析广叶绣球菌菌丝(Myc)、原基(Pri)和子实体(FB)生长阶段栽培基质中代谢产物的变化，通过不同数据库进行代谢物注释并进行KEGG通路富集分析。利用LC-MS/MS技术检测不同发育阶段绣球菌中植物类激素含量。【结果】三个不同栽培阶段基质中共鉴定出代谢产物1 360个。不同比较组(Pri vs. Myc、FB vs. Myc和FB vs. Pri)间共有的差异代谢产物179个，含量最高的50个代谢物主要包括氨基酸、脂质、吡喃酸、吡喃酮和植物类激素等物质。其中氨基酸含量在Myc、Pri和FB阶段基质中逐渐降低，而吡喃酸和吡喃酮类化合物含量逐渐升高。植物类激素中的赤霉素在Pri和FB阶段基质中含量较高，茉莉酸在Myc阶段基质中含量较高。对不同发育阶段绣球菌植物类激素进行检测，发现赤霉素GA7仅在原基中检测到，1...     

Plant growth regulators promote lipid and carotenoid accumulation in <Emphasis Type="Italic">Chlorella vulgaris</Emphasis>

Microalgae are photosynthetic organisms with the ability to produce a variety of high-value compounds such as polyunsaturated fatty acids (PUFAs), proteins, pigments, and lipids. The high cost of microalgae production is one of the biggest obstacles for their commercialization. Plant growth regulators might be an ideal choice since they could potentially induce microalgae to produce lipids and other high-value secondary metabolites thereby reducing production cost. This study investigated the effects of eight plant growth regulators (PGRs), namely, salicylic acid (SA); 1-naphthaleneacetic acid (NAA); gibberellin (GA₃); 6-benzylaminopurine (6-BA); 2,4-epibrassinolide (EBR); abscisic acid (ABA); ethephon (ETH); and spermidine (SPD) on the induction of lipids, proteins, carotenoids, and unsaturated fatty acids (UFAs) in Chlorella vulgaris. Moreover, the expression profiles of seven fatty acid biosynthethis genes were studied in the PGR-treated biomass. All PGRs used in the study caused significant increases in total lipid contents in non-dose-dependent manners when compared to control. However, lipid productivities were increased due to four of the eight PGRs (ABA, 6-BA, NAA, and ETH). Similar to lipids, total carotenoid contents were significantly higher in all of the PGR-treated microalgal biomass except ABA. However, soluble protein contents were not affected by the PGR treatments except SA at 10 mg L^?1. Furthermore, 6-BA, NAA, ABA, and ETH treatments resulted in significant increases in UFAs especially DHA, linolenic acid, arachidonic acid, and EPA which were confirmed by the upregulation of fatty acid biosynthesis genes including stearoyl-ACP-desaturase, ω-3 fatty acid desaturase, biotin carboxylase, and acyl-acyl carrier protein. Our findings, therefore, indicate that the treatment with PGR used in this study could be a useful tool to produce biodiesel and other high-value metabolites from microalgal biomass.     

A Comprehensive Search of the Primary and Secondary Metabolites and Radical Scavenging Potential of Trillium govanianum Wall. ex D. Don

Trillium govanianum rhizomes are traditionally consumed as a raw powder and decoction for the treatment of health complications. Hence, the present study aimed to investigate whether aqueous and alcoholic extracts of T. govanianum rhizomes under hot and cold extraction conditions have similar or dissimilar chemical, nutrient, and antioxidant profiles. The total phenolics, flavonoids, carbohydrates, proteins, fats, and energy values were estimated in all the conditionally prepared samples. The total phenolics (21.23±1.4 mg GAE/g extract), flavonoids (70.57±3.24 mg RE/g extract) were found higher in hot ethanolic extract (TGHEt), while cold water extract (TGGC) showed higher nutrients including amino acids (10.545±0.219 mg/g) and nucleosides (1.803±0.018 mg/g). The nutrient energy value (2.60 and 2.49 Kcal/g extract) was higher in cold and hot ethanolic extracts. Further, TGHEt scavenged the DPPH^. (IC₅₀; 870±22 μg/mL) and ABTS^.+ (IC₅₀; 80±1.49 μg/mL) effectively and proved its highest antioxidant activity compared to other samples. In LC/MS/MS-based metabolite profiling, twenty-six metabolites (fatty acids, steroidal saponins, triterpene saponins, ecdysteroid hormones) were confirmed with mass fragmentation and literature, while one hundred nine metabolites were identified using the METLIN database. The principal component analysis showed clustering of hot condition extracts while cold extracts were differentially located in quadrants. The heatmaps exhibited the associations and differences between metabolite composition, solvents, and extraction conditions. The identified metabolites speculatively predicted the biosynthesis pathway of T. govanianum. Findings also illustrated that T. govanianum is a source of bioactive nutritional components and saponins. The current metabolite profiling of T. govanianum will help in its agricultural and biotechnological interventions for higher quality produce.     

Long-chain fatty acids and ethanol affect the properties of membranes inDrosophila melanogaster larvae

The larval fatty acid composition of neutral lipids and membrane lipids was determined in three ethanol-tolerant strains ofDrosophila melanogaster. Dietary ethanol promoted a decrease in long-chain fatty acids in neutral lipids along with enhanced alcohol dehydrogenase (EC 1.1.1.1) activity in all of the strains. Dietary ethanol also increased the incorporation of¹⁴C-ethanol into fatty acid ethyl esters (FAEE) by two- to threefold and decreased the incorporation of¹⁴C-ethanol into free fatty acids (FFA). When cultured on sterile, defined media with stearic acid at 0 to 5 mM, stearic acid decreased ADH activity up to 33%. In strains not selected for superior tolerance to ethanol, dietary ethanol promoted a loss of long-chain fatty acids in membrane lipids. The loss of long-chain fatty acids in membranes was strongly correlated with increased fluidity in hydrophobic domains of mitochondrial membranes as determined by electron spin resonance and correlated with a loss of ethanol tolerance. In the ethanol-tolerant E2 strain, which had been exposed to ethanol for many generations, dietary ethanol failed to promote a loss of long-chain fatty acids in membrane lipids. We are grateful for the support of National Institutes of Health Grant AA06702 (B.W.G.) and National Science Foundation Grant CHE-891987 (R.G.K.).     

Oxalic acid production from lipids by a mutant of Aspergillus niger at different pH

Crude rapeseed oil and post-refining fatty acids were used as substrates for oxalic acid production by a mutant of Aspergillus niger. Both the final concentration and the yield of the product were highest at pH 4 to 5. With a medium containing 50 g lipids l^–1, production reached a maximum of 68 g oxalic acid l^–1 after 7 d. A high yield of the product (up to 1.4 g oxalic acid g^–1 lipids consumed) was achieved with oil and fatty acids combined.     

Effect of Conidiobolus coronatus on the Cuticular and Internal Lipid Composition of Tettigonia viridissima Males

Conidiobolus coronatus is an entomopathogenic fungus which has a potential as a biological control agent of insects. The cuticular and internal lipid composition of infected and noninfected Tettigonia viridissima males were analyzed by GC/MS. A total of 49 compounds were identified in the infected and noninfected males, including fatty acids, fatty acid methyl esters (FAMEs), n‐alkanes, alcohols, sterols, and other organic compounds. The most abundant components of the cuticular and internal lipids of the insects were fatty acids. After exposure to C. coronatus, the cuticular lipids of the T. viridissima males contained 17 free fatty acids from C(8) to C(22), while the cuticular lipids of the noninfected insects contained only 15 fatty acids from C(12) to C(24). The cuticular and internal lipids of both the infected and the noninfected males also contained five FAMEs from C(15) to C(19), seven n‐alkanes from C(25) to C(34), five alcohols from C(16) to C(25), five sterols, and the following six other organic compounds: azelaic acid, phenylacetic acid, glutaric acid, benzoic acid, sebacic acid, and glycerol. The compounds which were present only in the cuticular lipids of the infected males could be due to fungal infection.     

Analysis of Oxcarbazepine and the 10‐Hydroxycarbazepine Enantiomers in Plasma by LC‐MS/MS: Application in a Pharmacokinetic Study

Oxcarbazepine is a second‐generation antiepileptic drug indicated as monotherapy or adjunctive therapy in the treatment of partial seizures or generalized tonic–clonic seizures in adults and children. It undergoes rapid presystemic reduction with formation of the active metabolite 10‐hydroxycarbazepine (MHD), which has a chiral center at position 10, with the enantiomers (S)‐(+)‐ and R‐(?)‐MHD showing similar antiepileptic effects. This study presents the development and validation of a method of sequential analysis of oxcarbazepine and MHD enantiomers in plasma using liquid chromatography with tandem mass spectrometry (LC‐MS/MS). Aliquots of 100 μL of plasma were extracted with a mixture of methyl tert‐butyl ether: dichloromethane (2:1). The separation of oxcarbazepine and the MHD enantiomers was obtained on a chiral phase Chiralcel OD‐H column, using a mixture of hexane:ethanol:isopropanol (80:15:5, v/v/v) as mobile phase at a flow rate of 1.3 mL/min with a split ratio of 1:5, and quantification was performed by LC‐MS/MS. The limit of quantification was 12.5 ng oxcarbazepine and 31.25 ng of each MHD enantiomer/mL of plasma. The method was applied in the study of kinetic disposition of oxcarbazepine and the MHD enantiomers in the steady state after oral administration of 300 mg/12 h oxcarbazepine in a healthy volunteer. The maximum plasma concentration of oxcarbazepine was 1.2 µg/mL at 0.75 h. The kinetic disposition of MHD is enantioselective, with a higher proportion of the S‐(+)‐MHD enantiomer compared to R‐(?)‐MHD and an AUC^0‐12 _{S‐(+)/R‐(?)} ratio of 5.44. Chirality 25:897–903, 2013. © 2013 Wiley Periodicals, Inc.     

Effets physiologiques de ľ atrazine à doses sublétales sur Lemna minor, VII. Incorporation ďacétate-l,2-[14C] dans les groupes de lipides et leurs acides gras

Physiological effects of sublethal doses of atrazine on Lemna minor. VII. 1,2-[¹⁴C] acetate incorporation into the groups of lipids and their fatty acids. The lipids and the fatty acids of ten-day old duckweed (Lemna minor L.), cultivated aseptically in mineral solution containing sublethal concentrations of 0,10 and 0,50 ppm (0.46 and 2.3 μM, respectively) of atrazine, were analyzed by thin-layer chromatography and gas-liquid radiochromatography after 1,2-[¹⁴C] acetate feeding. Sublethal concentrations of atrazine increased the incorporation of radioactivity in total lipids, diacylgalactosylglycerol (DGG), diacyldigalactosylglycerol (DDG), sul-folipids (SL), phosphatidylglycerol (PG), diacylglycerol (DAG) and triacylglycerol + steroll esters (TAG+SE). The incorporation of acetate-1,2-[¹⁴C] decreased in phos-phatidylcholine (PC) and in phosphatidylethanolamine (PE) in the presence of atrazine. The radioactivity increased in total Transic-hexadecenoic, linoleic and α -linolenic acids while it decreased in the other fatty acids. This indicates that the sublethal concentrations of atrazine stimulate the desaturation of fatty acids of L. minor. The radioactivity was strongly incorporated in the α -linolenic acid of DGG in the presence of atrazine. The specific radioactivity of α-linolenic acid was greater in DAG than in PG > TAG + SE > PC > PE > DGG > SL > DDG and it increased in all groupd of lipids analyzed under the influence of sublethal doses of atrazine. The labelling of Translchexadecenoic acid of PG and its specific radioactivity increased in the presence of atrazine. These changes suggest that the sublethal concentrations of atrazine stimulate especially the lipid metabolism of the chloroplasts of L. minor and they could explain the increase in the number of grana per chloroplast in treated L. minor. The results are discussed in relation to the biosynthesis of galactolipids.     

Quantification of nitrite and nitrate in human urine and plasma as pentafluorobenzyl derivatives by gas chromatography—mass spectrometry using their N-labelled analogs

For the quantification of nitrite and nitrate, the stable metabolites of -arginine-derived nitric oxide (NO) in human urine and plasma, we developed a gas chromatographic—mass spectrometric (GC—MS) method in which [¹⁵N]nitrite and [¹⁵N]nitrate were used as internal standards. Endogenous nitrite and [¹⁵N]nitrite added to acetone-treated plasma and urine samples were converted into their pentafluorobenzyl (PFB) derivatives using PFB bromide as the alkylating agent. For the analysis of endogenous nitrate and [¹⁵N]nitrate they were reduced to nitrite and [¹⁵N]nitrite, respectively, by cadmium in acidified plasma and urine samples prior to PFB alkylation. Reaction products were extracted with toluene and 1-μl aliquots were analyzed by selected-ion monitoring at m/z 46 for endogenous nitrite (nitrate) and m/z 47 for [¹⁵N]nitrite ([¹⁵N]nitrate). The intra- and inter-assay relative standard deviations for the determination of nitrite and nitrate in urine and plasma were below 3.8%. The detection limit of the method was 22 fmol of nitrite. Healthy subjects (n = 12) excreted into urine 0.49 ± 0.25 of nitrite and 109.5 ± 61.7 of nitrate (mean ± S.D., μmol/mmol creatinine) with a mean 24-h output of 5.7 μmol for nitrite and 1226 μmol for nitrate. The concentrations of nitrite and nitrate in the plasma of these volunteers were determined to be (mean ± S.D., μmol/l) 3.6 ± 0.8 and 68 ± 17, respectively.     

Isolation and characterization of a long-chain acyl-coenzyme A synthetase encoding gene from the marine microalga Nannochloropsis oculata

Acyl-coenzyme A synthetases (ACSs) are associated with the anabolism and catabolism of fatty acids and play fundamental roles in various metabolic pathways. The cDNA of long-chain acyl-coenzyme A synthetase (LACS), one of the ACSs, was isolated from Nannochloropsis oculata and named as NOLACS. The predicted amino acid sequence was highly similar to LACSs of other species. NOLACS encodes a long-chain acyl-coenzyme A synthetase; it recovered the function of LACS in Saccharomyces cerevisiae YB525 (a LACS-deficient yeast strain). The substrate specificity of the enzyme was also assayed in yeast. It was found that NOLACS can activate saturated fatty acids (C12:0, C14:0, C16:0, and C18:0) and some unsaturated fatty acids (C18:2Δ^{9, 12} and C20:2Δ^{11, 14}) with a preference for long-chain fatty acids. Our findings will provide a deep understanding of CoA-dependent fatty acid activation and also make some contribution to understanding the metabolic pathways of lipids in Nannochloropsis. These findings will also facilitate studies on the regulation of gene expression and genetic modification of fatty acid synthesis and storage of N. oculata.     

Metabolomics Analysis of Metabolites in <i>Forsythia suspense</i> Fruit Using UPLC/ESI-Q TRAP-MS/MS

Forsythiae Fructus, the fruit of Forsythia suspense is a traditional Chinese hebal medicine that has the antiviral and antioxidant effects in China. Modern analytical chemistry studies showed that the extracts of Forsythiae Fructus contain many bioactive components, such as flavonoids, lignans, phenolic acids, and terpenoids, which can be used to anti-inflammatory and treat toxicity, tonsillitis, ulcers, pharyngitis and acute nephritis. In order to study the types and quantities of metabolites in Forsythiae Fructus, we isolated, identified and analysed metabolites between two varieties of Forsythiae Fructus using UPLC/ESI-Q TRAP-MS/MS. The results showed that a total of 407 metabolites were identified in Forsythiae Fructus using UPLC/ESI-Q TRAP-MS/MS, including 21 terpenoids, 68 phenolic acids, 63 flavonoids, 43 amino acids and derivatives, 22 alkaloids, 55 lipids, 24 lignans and coumarins, 31 nucleotides and derivatives, 29 organic acids, and 51 other metabolites. Among, lignans and coumarins, terpenoids, organic acids, lipids, and phenolic acids were rich in Forsythiae Fructus, which accounted for more than 60% of the total metabolite content. Differential metabolite analysis revealed that 80 metabolites differed significantly between the two types of Forsythiae Fructus. Our results greatly enrich the Forsythiae Fructus phytochemical composition database and provide valuable information for further study of the metabolites of Forsythiae Fructus.     

Enantiospecific Analysis of 8‐Prenylnaringenin in Biological Fluids by Liquid‐Chromatography‐Electrospray Ionization Mass Spectrometry: Application to Preclinical Pharmacokinetic Investigations

8‐Prenylnaringenin (8PN) is a naturally occurring bioactive chiral prenylflavonoid found most commonly in the female flowers of hops (Humulus lupulus L.). A stereospecific method of analysis for 8PN in biological fluids is necessary to study the pharmacokinetic disposition of each enantiomer. A novel and simple liquid chromatographic‐electrospray ionization‐mass spectrometry (LC‐ESI‐MS) method was developed for the simultaneous determination of R‐ and S‐8PN in rat serum and urine. Carbamazepine was used as the internal standard (IS). Enantiomeric resolution of 8PN was achieved on a Chiralpak^® AD‐RH column with an isocratic mobile phase consisting of 2‐propanol and 10 mM ammonium formate (pH 8.5) (40:60, v/v) and a flow rate of 0.7 mL/min. Detection was achieved using negative selective ion monitoring (SIM) of 8PN at m/z 339.15 for both enantiomers and positive SIM m/z at 237.15 for the IS. The calibration curves for urine were linear over a range of 0.01–75 µg/mL and 0.05–75 µg/mL for serum with a limit of quantification of 0.05 µg/mL in serum and 0.01 µg/mL in urine. The method was successfully validated showing that it was sensitive, reproducible, and accurate for enantiospecific quantification of 8PN in biological matrices. The assay was successfully applied to a preliminary study of 8PN enantiomers in rat. Chirality 26:419–426, 2014. © 2014 Wiley Periodicals, Inc.     

Gas chromatography–mass spectrometric method for quantitative determination in human urine of dicarboxylic (dioic) acids produced in the body as a consequence of cholesterol biosynthesis inhibition

A capillary gas chromatography–mass spectrometric (GC–MS) method in human urine has been developed and validated for the quantitative determination of dicarboxylic acids (dioic acids) which are produced in the body as a consequence of the administration of an inhibitor of the enzyme squalene synthase, which is involved in the biosynthesis of cholesterol. The standards and quality control (QC) samples were prepared by adding dioic acids into human urine. Internal standard (sebacic acid) was added to each urine sample (0.1 ml) and then dried by evaporation under nitrogen. The dried sample was reacted with pentafluorobenzyl (PFB) bromide under conditions that maximized the formation of the di-PFB ester (at the expense of the mono-PFB ester) of the dioic acids. After drying by evaporation, each sample residue was reconstituted in mesitylene and injected into a capillary GC–MS system via a splitless injection. The detection was by negative ion chemical ionization mass spectrometry with selected ion monitoring (SIM) of the [M−PFB]⁻ of the analytes and the internal standard.     

Physiological Status and Community Composition of Microbial Mats of the Ebro Delta, Spain, by Signature Lipid Biomarkers

Abstract Physiological status of microbial mats of the Ebro Delta (Tarragona, Spain) based on the extraction of lipids considered ``signature lipid biomarkers' (SLB) from the cell membranes and walls of microorganisms has been analyzed. Data from a day–night cycle show significant differences in viable cells countings (PLFA cells counts) ranging from 1.5 × 10¹⁰ to 5.0 × 10¹⁰ cells g⁻¹ of sediment. Minimum values were observed at 18:00 and 6:00, when physicochemical conditions change drastically. The diversity of the microbial community was assessed by GC/MS analysis of phospholipid fatty acids (PLFA). The ratio of PLFA, representative of Gram-negative bacteria, comprises 47.8% of the total PLFA of the microbial mat community. The remaining PLFA was representative of Gram-positive (10.0%), anaerobic (5.7%), and eukaryotic microorganisms (5.7%), and other common lipids. Two different approaches were used as a comparative study to assess the physiological status of the microbial mats. Two parameters (cyclopropane fatty acids/ω7c monoenoic fatty acids, and measurement of the trans/cis monoenoic PLFA ratio) showed a minimum at midnight, suggesting the highest microbial activity. Higher values were observed at 18:00 and 6:00, coinciding with lower PLFA cell counts. Received: 14 May 1999; Accepted: 6 September 1999; Online Publication: 24 March 2000     

RADIOLABELING STUDIES OF LIPIDS AND FATTY ACIDS IN NANNOCHLOROPSIS (EUSTIGMATOPHYCEAE), AN OLEAGINOUS MARINE ALGA1

The synthesis of fatty acids and lipids in Nannochloropsis sp. was investigated by labeling cells in vivo with [¹⁴C]-bicarbonate or [¹⁴C]-acetate. [¹⁴C]-bicarbonate was incorporated to the greatest extent into 16:0, 16:1, and 14:0 fatty acids, which are the predominant fatty acids of triacylglycerols. However, more than half of the [¹⁴C]-acetate was incorporated into longer and more desaturated fatty acids, which are constituents of membrane lipids. [¹⁴C]-acetate was incorporated most strongly into phosphatidylcholine, which rapidly lost label during a 5-h chase period. The label associated with phosphatidylethanolamine also decreased during the chase period, whereas label in other membrane lipids and triacylglycerol increased. The dynamics of labeling, along with information regarding the acyl compositions of various lipids, suggests that 1) the primary products of chloroplast fatty acid synthesis are 14:0, 16:0, and 16:1; 2) C₂₀ fatty acids are formed by an elongation reaction that can utilize externally supplied acetate; 3) phosphatidylcholine is a site for desaturation of C₁₈ fatty acids; and 4) phosphatidylethanolamine may be a site for desaturation of C₂₀ fatty acids.